| Research backgroundAccording to whether there are double bonds in the structure of the carbon chain,fatty acids are divided into saturated fatty acids and unsaturated fatty acids.The difference between saturated fatty acids lies in the hydrocarbon chain length of their structure.In addition to the Carbone chain difference,the differences between unsaturated fatty acids are different locations and amounts of double bounds as well.Carbon chain of unsaturated fatty acids contains more than 1 or 1 double bonds.According to the quantities of double bonds,unsaturated fatty acid with 1 double bond is called mono-Unsaturated fatty acids,and the unsaturated fatty acids with 2 or more than two double bonds is refereed to polyunsaturated fatty acids,PUFAs.It consists of omega-3,omega-6 and omega-9 series compared to the atom location of the last double bonds from carboxy-terminal.Most important of all,omega-3 and omega-6 series of PUFAs are essential fatty acids of the body and the major constituent of the membrane phospholipids.They are not synthesized by the body and must be obtained from the diet.That Omega-3 fatty acids(ω-3 fatty acids or n-3 fatty acids)is long-chain polyunsaturated fatty acids consists of 18-22 carton atoms including Alpha-Linolenic acid(ALA)、Docosahexaenoic(DHA)and Eicosapentaenoic(EPA).In the 1980s,Danish scientists initially discovered and reported that the Eskimos(Inuits)live in the Arctic who take abyssal fishes as the diet,which has higher content of unsaturated fatty acid and cholesterol,but incidence of Coronary Heart Disease among Eskimos are lower.Subsequent analyses indicated that there is relevant between diet rich in omega-3 PUFAs and the lower risks of cardiovascular disease(CVD).It can reduce coronary heart disease morbidity and mortality remarkably.With developing of the research,more positive health effects of omega-3 PUFAs have been found,which relates to many fields,such as cardiovascular disease,hypertension,diabetes,hyperlipidemia and obesity and other metabolic diseases.Meanwhile,it plays positive roles of anti-tumor,anti-depression,diminishing inflammation,and facilitates the growth of brain,retina,skeleton and developments.The content of omega-3 PUFAs in the body can be adjusted by dieting.However,it’s hard to exactly calculate the content of omega-3 PUFAs in the diet because different foods contain different levels of omega-3 PUFAs.Furthermore,tissue and cellular metabolism in the body are different.Therefore,formulating an objective indicator to measure the content and distribution of omega-3 PUFAs is necessary.In 2004,Harris and Von Schacky initially put forward a concept of omega-3(ω-3 or n-3)index,which refers to the proportion of EPA and DHA in the erythrocyte membrane,and it is free from diet in a short period,and reflects long term(one or two month)dietary patterns and nutrition levels of fatty acids of the body.Many research results indicate that there is a significant negative correlation between omega-3 and risk and mortality of coronary heart disease(CHD),which is valuable to clinical evaluation of CHD,and it has become a new factor for evaluating the risks of CHD.It has preventive effects on cardiovascular disease,and takes lower incidence risks when the omega-3 index is greater than or equal to 8%.However,it takes higher risks of cardiovascular diseases,and its protective effect on heart is weak when omega-3 index is less than 4%.Risk of sudden cardiac death is 10 times when the omega-3 index is greater than 8%.Higher omega-3 index(Greater than or equal to 8%)can be one of the clinical treatment objectives of the cardiovascular diseases.Omega-3 PUFAs distributes in triglycerides,cholesterol esters,erythrocyte membrane and all kinds of fatty tissues.At present,there are many omega-3 PUFAs evaluation methods in the epidemiological survey and clinical research at home and abroad,such as dietary intake questionnaire,plasma phospholipid fatty acid profile,omega-3 PUFAs indicator of plasma cholesterol lipid,omega-3 index of erythrocyte membrane and other methods.The current determination methods at home and abroad are to detect the fatty acid components in the plasma or serum.Generating the silanizing or transesterification product by deriving silane or esterification of fatty acids is the major pre-processing method.The current detection methods include high-performance liquid chromatography(HPLC),gas chromatography(GC),liquid chromatography-tandem mass spectrometry and other methods.Nevertheless,study on determination methods of fatty acid of erythrocyte membrane is rare relatively.Most of them are GC detection method.At present,there are hundreds of fatty acids separated from organisms,and most structure and properties of fatty acids are similar.It is prone to cause quantitatively inaccurate of classifications if the compound of samples are merely separated and detected with GC and lack of qualitative steps.On the other hand,GC detection method is difficult to adapt to the rapid test and screening larger population because its operation is complex and need longer time inthe detection.Therefore,it is particularly important to establish a rapid,sensitive,accurate and specific method for the detection of erythrocyte fatty acids.Components of fatty acids in the erythrocyte can reflect the long-term components of diet of the crowd.It is not affected by dietary factors in a short-term,and the measured contents of fatty acids can reflect the different nutritional levels of fatty acids in the body objectively.Therefore,it can provide an objective basis for adjusting diet structure of the body and increasing the intake of unsaturated fatty acids.Especially the intake of the omega-3,it can change the composition of fatty acids in the erythrocyte membrane,which is beneficial to health.Methods1.It takes erythrocyte separated from EDTA whole blood as the sample,and conducts esterification after adding fatty acids IS and HCl methanol into the sample,and then,it is to conduct liquid-liquid extraction with n-hexane,and transfers the upper solution after extracting,and dries it up with nitrogen,and dissolves it again by adding n-hexane.Finally,the complex solution is detected with gas chromatograph-mass spectrometer.Various Fatty Acid Composition and Standard parameters of the samples are compared with the standard spectrum library,and then,it is saved as the qualitative evidence.The fatty acid composition of erythrocyte membrane is analyzed by internal standard curve method and quantitative analysis.The established methodology is appraised from many aspects of the detection method,such as accuracy,precision,linear range,analysis of sensitivity,stability of the sample and other angles.2.A total of 149 healthy objects from Guangzhou area were enrolled,and objects’erythrocyte fatty acid composition and omega-3 index were examined with the established method above for observing subjects’ distribution of fatty acid composition of erythrocyte and distribution interval of omega-3 index.3.Totally 29 objects and 22 patients with primary hypertension aged 40-60 from Guangzhou area were enrolled as the objects of study.Objects’ fatty acid composition of erythrocyte distribution was detected with the established GC-MS above.Indicators such as omega-3 were calculated as well,and then,the two groups’parameters,such as distribution of fatty acid of erythrocyte were compared.Results1.Analytical condition for the erythrocyte fatty acids of GC-MS detection:①Chromatographic condition:adopts Agilent DB-23 capillary column(60m×0.25mm×0.15um);sampling model irrespective,injection volume 1μL,chromatographic column flow 0.78mL/min;injection orifice temperature:230℃;interface temperature:250℃;initial column temperature:50℃.Heats up to 223 ℃ with the speed of 4℃/min and keeps it for 8 minutes,and then,it is heated to 250℃ with the speed of 20℃/min and holds for 12 minutes;② Mass spectrum analysis condition:adopts El model.Ionization source temperature:220℃;solvent delay time:10 minutes;scan model:full scan and SIM;full scan range:m/z 60.00-450.00.Fatty acid standards and internal standards can be distinguished easily with the established methods,and the detection process can be done in 12 minutes.2.Method validation shows that erythrocyte fatty acid detection method established in this study is sensitive,and its minimum detection limit reached O.Olmg/L;Linearity of the detection is well.Linear range of detection:0.02-407.04mg/L.Inter-batch and within-batch precision of the omega-3 meet the demand.Compared with the traditional detection methods,this method for the detection of erythrocyte fatty acid has higher flux,and needs shorter time.3.In this study,more than 20 kinds of fatty acids in the 149 objects’ erythrocyte membrane account for 0.03%-22.44%in the total fatty acid content,and the minimum accounts for C18:3n6,and the maximum accounts for C20:4n6;monounsaturated fatty acid content accounts for 19.29%;omega-3 series among polyunsaturated fatty acid accounts for 7.24%,omega-6 accounts for 39.29%;mean value of erythrocyte omega-3 index accounts for 5.82%,in which 95%distribution interval is between 3.59-8.44%.4.The group with primary hypertension and the health group’s C22:5n3(P=0.018),C20:4n6(P =0.042),total omega-6 fatty acids:(p=0.028),omega-6/omega-3(p=0.007)and AA/EPA(p=0.005).There was significant difference,and there was no significant difference of other indicators.Conclusions1.A method of testing fatty acid composition of erythrocyte membrane with GC-MS is established.Its preprocessing is easy,sensitive,precise,specific,and it has stable methodology.It needs shorter time to detect,and it is applicable to rapid clinical detection and mass screening.2.Fatty acid composition and distribution and omega-3 index of erythrocyte of the 149 healthy objects from Guangzhou area were analyzed,and the fatty acid composition and omega-3 reference interval of erythrocyte of the crowd were given.3.The total Omega-6 fatty acids,Omega-6/Omega-3 and AA/EPA in erythrocyte of primary hypertension crowd are higher than these in healthy people. |
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